EP0176080B1 - Blood partitioning method and apparatus - Google Patents

Blood partitioning method and apparatus Download PDF

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Publication number
EP0176080B1
EP0176080B1 EP85112104A EP85112104A EP0176080B1 EP 0176080 B1 EP0176080 B1 EP 0176080B1 EP 85112104 A EP85112104 A EP 85112104A EP 85112104 A EP85112104 A EP 85112104A EP 0176080 B1 EP0176080 B1 EP 0176080B1
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EP
European Patent Office
Prior art keywords
gel
substance
accordance
density gradient
specific gravity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
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EP85112104A
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German (de)
English (en)
French (fr)
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EP0176080A2 (en
EP0176080A3 (en
Inventor
Alex M. Saunders
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Becton Dickinson and Co
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Becton Dickinson and Co
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Application filed by Becton Dickinson and Co filed Critical Becton Dickinson and Co
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Publication of EP0176080A3 publication Critical patent/EP0176080A3/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D43/00—Separating particles from liquids, or liquids from solids, otherwise than by sedimentation or filtration
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Definitions

  • the present invention relates generally to a method and apparatus for partitioning a heavier phase from a lighter phase of a centrifugally separated multiphase fluid specimen. More particularly, the present invention is directed to a method and apparatus for separating platelets, lymphocytes and monocytes from granulocytes and erythrocytes in a blood specimen.
  • the Immunological analysis of blood generally requires the isolation and separation of the lymphocytes for detailed analysis.
  • two distinct methods are known for the separation of lymphocytes from other blood cells.
  • the first of these methods involves buoyant density centrifugation of cells through a particular newtonian fluid.
  • the most commonly used fluid is known as Ficoll-PaqueTM, a water soluble liquid having a specific gravity of 1.077 g/cm 3 , which is marketed by Pharmacia Fine Chemicals AB, Upsala, Sweden.
  • the second of these methods utilizes a non-newtonian, water-insoluble, thixotropic, gel-like substance which establishes a continuous semi-rigid gel-like seal across the interior of a container between the lighter phase containing the lymphocytes and a heavier phase containing the erythrocytes and granulocytes.
  • the Ficoll-PaqueTM method contemplates the following general steps:
  • the Ficoll-PaqueTM method has several disadvantages. First, very careful technique by the operator of the Ficoll-PaqueTm method is required. If the initial introduction of the blood sample is performed carelessly, plasma may be deployed below the surface of the Ficoll-PaqueTM medium causing reduced local specific gravity of the Ficoll-PaqueTM which is then inadequate to separate lymphocytes and monocytes from other cells.
  • centrifugation forces higher than 400 g cannot be utilized since the Ficoll-PaqueTM is water soluble and higher centrifugation forces dilution of the Ficoll-PaqueTM with the blood plasma, thereby resulting in a change in the Ficoll-PaqueT" specific gravity and a substantial alteration in the separation efficiency.
  • a more rapid process is provided by methods which utilize a non-newtonian water immiscible, thixotropic, gel-like substance (hereinafter referred to as a "gel-like substance") for establishing a barrier between the lighter phase containing the lymphocytes and a heavier phase containing the erythrocytes and granulocytes.
  • This method is exemplified by United States Patent Number 4,190,535 to Luderer; United States Patent Number 3,852,194 to Zine; United States Patent Number 4,147,628 to Bennett; United States Patent Number 4,350,593 to Kessler; and United States Patent Number 4,153,739 to Kessler.
  • the use of a gel-like substance to establish a barrier layer between the lighter phase and the heavier phase generally provides a more rapid and easier method for separating the lighter phase and heavier phase than the Ficoll-PaqueTM method.
  • the use of the gel-like substance in a blood separation scheme also has disadvantages. The most important disadvantage is that the lymphocyte layer which lies immediately above the gel-like substance after the centrifugation step tends to become contaminated with granulocytes which lie immediately below the gel-like substance.
  • the present invention is directed to a method for separating lymphocytes and monocytes from erythrocytes and granulocytes in a blood sample which is more rapid than the Ficoll-Paque"' method and which provides a better separation than the methods utilizing a thixotropic gel-like material.
  • the method of the present invention contemplates the following general steps:
  • An important feature of the present invention is that, as the heavier cells of the blood pass through the water soluble density gradient material, they carry with them some plasma from the blood. This dilutes a portion of the water soluble density gradient material which then passes during centrifugation through or around the gel-like substance to form an intermediate water soluble density gradient layer above the gel-like substance and beneath the mononuclear cells.
  • This intermediate layer of water soluble density gradient material is effective, in combination with the substantial mass of the water soluble density gradient material lying beneath the layer of gel-like substance to effectively isolate the granulocytes from contamination of the mononuclear layer of cells. This provides a cleaner more effective separation of the mononuclear cells without being contaminated with granulocytes.
  • the thin layer of water soluble density gradient material provides a cushion support for the mononuclear cell layer during centrifugation which prevents tight packing of the layer of mononuclear cells.
  • the formulation or composition of the gel-like substance is not critical. Any of the prior art compositions describing water immiscible, thixotropic gel-like materials can be used in the method of the present invention.
  • United States Patent No. 4,190,535 describes the use of a mixture of silicone fluid and very fine hydrophobic silica powder to provide a gel-like substance.
  • a hydrocarbon gel-like material polybutane 11-100, marketed by Amoco Chemicals Corporation, Chicago, Illinois and described in that company's bulletin 12-11 as a butylene polymer composed predominantly of high molecular weight mono-olefins (85-98%), the balance being isoparaffins.
  • the polybutylene is mixed with fumed silica powder to provide a water-immiscible, thixotropic gel-like material.
  • fumed silica powder is also useful.
  • a hydroxyl terminated homopolymer of butadiene with the degree of polymerization being about 50.
  • fumed silica powder is mixed with fumed silica powder to provide a suitable water-immiscible thixotropic, gel-like material.
  • a preferred material for use as the gel-like substance is a polyester, such as that described in United States Patent No. 4,101,422 to Lamont et al. Particularly preferred is a single component polyester #NB 2042-108, a polyester manufactured by Emery Industries, Inc.
  • the polyester composition is particularly preferred for use as the gel-like substance in the present invention since it does not need to be combined with silica powder to provide a suitable specific gravity. It has been determined that the use of silica powder is detrimental to analysis of the recovered lymphocyte fraction in certain apparatus, such as flow cytometry devices. Therefore, the use of a polyester having a suitable specific gravity is preferred.
  • the gel-like substance should meet the following criteria:
  • the water soluble density gradient material is preferably a solution of a polymeric saccharide and a viscosity and density modifying substance.
  • the water-soluble density gradient material is most preferably a Ficoll-PaqueTM type of material.
  • Ficoll-Paque is an aqueous solution of Ficoll-400TM and diatrizoate sodium.
  • Ficoll-400TM is a synthetic high molecular weight (M w 4000) polymer of sucrose and epichlorohydrin which is easily soluble in water.
  • the molecules of Ficoll-400TM are highly branched, approximately spherical and have a low intrinsic viscosity compared with linear polysaccharides of the same molecular weight.
  • Diatrizoate sodium is a convenient compound to use with Ficoll-400TM since it forms solutions of low viscosity and high density.
  • Diatrizoate sodium (molecular weight 635.92) is the sodium salt of 3,5-diacetamido 2,4,6-triiodo benzoic acid.
  • the specific gravity of the density gradient material can be adjusted by varying the amounts of diatrizoate sodium contained in the aqueous solution of Ficoll-400TM.
  • Ficoll-PaqueTM contains 5.7 g Ficoll-400 and 9 g diatrizoate sodium in each 100 ml.
  • the specific gravity of the water soluble density gradient material is preferred to within the range of from 1.08 to 1.100 g/cm 3 , preferably from 1.085 to 1.095, most preferably 1.09 gm/ cm 3 .
  • an aqueous solution containing from 5.7 g to 6.0 g of sucrose polymer and from 11.0 g to 12.0 g of diatrizoate sodium is suitable for use as the water soluble density gradient material.
  • the method of the present invention is useful with both the conventional open blood collection tube and the closed blood collection tube wherein the opening is closed by a septum penetrable by a needle for insertion of a blood sample.
  • the gel-like substance and the water soluble density gradient material are placed into the tube in a suitable amount in no particular order.
  • a suitable amount of gel-like substance is about 1.2 grams and a suitable amount of the water soluble density gradient material is 1.0 grams. This wil permit the separation of blood samples of up to 5 ml.
  • the blood collection tube containing the gel-like substance and the water soluble density gradient material may be pre-centrifuged prior to the addition of a blood specimen.
  • Pre-centrifugation causes the water soluble density gradient material to migrate to the bottom of the tube and causes the gel-like substance to form a barrier over the top of the water soluble density gradient material.
  • the blood specimen can then be inserted into the blood collection tube without regard to careful technique.
  • Moderate centrifugation forces of from 50 g to 1,000 g for a period of from 5 minutes to 0.5 minutes, respectively, are suitable for the pre-centrifugation step.
  • the blood collection tube is centrifuged at a suitable force for a suitable time.
  • a centrifugation force of from 600 g to about 2000 g is used for a time of from 20 to 5 minutes, respectively.
  • the gel-like substance 11 forms a barrier atop the water soluble density gradient material 13 in blood collection tube 15.
  • a blood specimen 17 is placed atop the barrier of gel-like substance 11, as shown in Figure 2.
  • the erythrocytes 19 form the bottommost layer.
  • a granulocyte layer 21 lies atop the erythrocytes 19.
  • a major fraction of the water soluble density gradient material lies above the granulocyte layer 21.
  • the erythrocytes carry a small part of the blood plasma with them as they migrate through the layer of water soluble density gradient material.
  • the plasma fraction of the blood is then removed in a manner designed to leave the layer 25 of platelets, lymphocytes and monocytes intact on the surface of the layer 23 of water-soluble density gradient material.
  • the use of a Pasteur pipette is an example of a suitable manner for removing the plasma layer 27, leaving behind an undisturbed layer 25 of lymphocytes, monocytes and platelets.
  • Another example is the use of a syphon tube attached to a vacuum source.
  • the layer 25 of lymphocytes, monocytes and platelets can then be removed by either of two methods.
  • a Pasteur pipette is used to transfer the layer 25 to a clean centrifuge tube for washing.
  • a buffered saline solution can be poured directly onto the top of the layer 25 of mononuclear cells and platelets.
  • the barrier layer 11 of gel-like substance prevents interaction of the buffered saline solution with the granulocytes and erythrocytes.
  • the mononuclear cells can then be removed from atop the barrier layer 11 by simply pouring the mixture of buffered saline solution and mononuclear cells from the collection tube 16.
  • a sample of whole blood was taken from a single patient. The same blood sample was used for all of the subsequent examples. An aliquot of the blood sample was first used in a standard Ficoll-PaqueTM density gradient method to establish a control value. The density gradient method was performed as described in a brochure prepared by Pharmacia Fine Chemicals entitled Ficoll-PaqueTM Forln Vitro Isolation of Lymphocytes. The blood was treated by an anticoagulation agent, EDTA, prior to use in any of the examples.
  • EDTA anticoagulation agent
  • the yield obtained using the Ficoll-PaqueTM method was 90% of the mononuclear cells present in the blood sample.
  • the absolute value of the mononuclear cells present in the blood sample was determined by standard microscopic stain analysis.
  • the granulocyte level present in the mononuclear cells was 0.4%.
  • Example 1 The method of Example 1 was repeated using an aliquot of the blood sample which was diluted with an equal volume of buffered, isotonic salt solution (2 ml blood sample+2 ml buffered salt solution). The yield of mononuclear cells was 64% of the theoretical yield and the granulocyte content was 3.25%.
  • the method of the present invention was used to isolate the mononuclear cells in an aliquot of the blood sample.
  • 1.2 grams of a thixotropic, polyester gel having a density of 1.077 grams/ml was placed in a 7 ml test tube.
  • 1.0 grams of a density gradient material containing 5.8 grams of sucrose polymer and 10 grams of diatrizoate sodium dissolved in water was placed in the test tube.
  • the test tube was subjected to pre-centrifugation so as to form a barrier layer of the gel material above the density gradient material.
  • An aliquot of the blood sample was placed on top of the gel barrier layer. Centrifugation was effected at 900xG for 10 minutes.
  • the cells were harvested after syphoning off the plasma by adding buffered saline and providing gentle turbulence to the cells with a bulb and pipette.
  • the yield of mononuclear cells was 80% and the granulocyte content was 1.65%.
  • Example 3 The procedure of Example 3 was repeated with the exception that the aliquot of the blood sample was diluted with.an equal volume of buffered, isotonic salt solution. The yield of mononuclear cells was 60% and the granulocyte content was 1.94%.
  • Example 3 The procedure of Example 3 was used in comparison with the Ficoll-Paque 7 , density gradient method on blood samples extracted from 5 separate subjects.
  • Table I hereinbelow, illustrates the average yield and times required to perform the analysis:

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EP85112104A 1984-09-24 1985-09-24 Blood partitioning method and apparatus Expired - Lifetime EP0176080B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US653178 1984-09-24
US06/653,178 US4751001A (en) 1984-09-24 1984-09-24 Blood partitioning apparatus

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EP0176080A2 EP0176080A2 (en) 1986-04-02
EP0176080A3 EP0176080A3 (en) 1987-08-19
EP0176080B1 true EP0176080B1 (en) 1990-02-28

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US (1) US4751001A (enrdf_load_stackoverflow)
EP (1) EP0176080B1 (enrdf_load_stackoverflow)
JP (1) JPS6184557A (enrdf_load_stackoverflow)
DE (1) DE3576210D1 (enrdf_load_stackoverflow)

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JPS6184557A (ja) 1986-04-30
DE3576210D1 (de) 1990-04-05
US4751001A (en) 1988-06-14
EP0176080A2 (en) 1986-04-02
JPH0465981B2 (enrdf_load_stackoverflow) 1992-10-21
EP0176080A3 (en) 1987-08-19

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